The goal of this project is to elucidate the role of the ?7- nicotinic acetylcholine receptor (nAChR) and its associated proteome (i.e., interactome) in the pathophysiology of Alzheimer's Disease (AD) as a vehicle towards developing more targeted and efficacious treatments. We hypothesize that the regulatory and signaling proteins closely associated in vivo with hippocampal ?7-nAChRs play an important role in normal neuronal functions including those critical to memory, and that the signaling machinery of the ?7 macromolecular complex is adversely affected as AD progresses resulting in changes in the protein interactome of ?7-nAChRs. Using high-throughput proteomic technologies and post mortem tissue, we will focus on the cytoplasmic proteins associated with the ?7-nAChR, which constitutes the second most abundant nicotinic receptor system in the human brain. In Aim 1, we will determine the protein interacting partners of the ?7-nAChR in post mortem hippocampal tissue of aged subjects (70-75 years) with no history of AD. The ?7-nAChR and its interacting proteins will be isolated from the homogenate by ligand affinity pulldown (?-bungarotoxin[Bgtx]-Sepharose beads). 500 ?M methyllycaconitine (MLA) will be added to control homogenates to block selectively the binding of ?7-nAChR to the Bgtx-Sepharose beads. Bound proteins will be eluted with carbamylcholine, fractionated by SDS-PAGE, and tryptic digests prepared. The peptide identities will be determined using state-of-the-art mass spectrometric methods, and then proteins identified in experimental and control samples will be compared to filter out those that bind in a nonspecific fashion. Hippocampal tissue from at least 20 donors will be characterized in order to probe the population variation among interacting proteins associated with ?7-nAChR. These results will serve as the normotypic baseline for studies of pathological tissue. In Aim 2, we will determine how the composition of ?7-nAChR interacting proteins is altered in the post mortem hippocampal tissue of individuals with late stage AD. The proteomic data from AD samples will be compared to the proteomic data from Aim 1 to allow detailed analysis of the effects of AD on the protein interactors of the ?7-nAChR. In Aim 3, we will use label-free quantitative mass spectrometry on data collected in Specific Aims 1 and 2 to determine how the relative levels of proteins that interact with the ?7-nAChR are altered by AD. The mass spectrometry data collected in Aims 1 and 2 will be re-analyzed bioinformatically to quantify changes in ?7-nAChR associated proteins. The mass spectrometry data from a hybrid LTQ-Orbitrap Velos ETD instrument would generate data from which both identifying and quantitative conclusions could be drawn. The data from Aims 1 and 2 would be compared to determine alterations in protein levels caused by the pathophysiological mechanisms of AD. PUBLIC HEALTH RELEVANCE: Using a high-throughput technology and human post mortem tissue, we will study how cellular proteins found in close working association with an important human brain receptor are affected in Alzheimer's disease. We will pursue a proteomic approach to determine whether changes in the composition and function of the proteins found associated with nicotinic alpha7 receptors can be correlated with the disease. The results from our proposed studies should lead to a better understanding of the role of nicotinic alpha7 receptors in Alzheimer's disease. Future detailed study of the proteins to be identified in the proposed work could lead to the development of more highly targeted therapies for this disease.